1. Field of the Invention
The present invention relates generally to the fields of molecular biology and nucleic acid analysis. More specifically, the present invention relates to a novel method of genetic analysis using arbitrary sequence oligonucleotide fingerprinting.
2. Description of the Related Art
A certain amount of DNA sequence variation occurs naturally within a population of individuals. At many chromosomal positions, the frequency of sequence variation within a population is great enough to yield useful DNA markers, and the occurrence of a polymorphic allele at a frequency of about 10% is generally considered useful for mapping purposes (1). Analysis of DNA polymorphisms has been extremely valuable for identifying genetic markers tightly linked to genes associated with phenotypic traits. The use of gel electrophoresis to detect restriction fragment length polymorphism (RFLP) has yielded thousands of mapped polymorphic DNA markers in various species. The most frequent type of genetic change associated with an RFLP marker is point mutation within the recognition sequence of a restriction enzyme.
Although restriction fragment length polymorphism analysis remains a widely used method for detecting DNA sequence polymorphism, several useful variations on the fragment length theme have recently been introduced. The existence of variable number tandem repeat (VNTR) or "microsatellite" sequences scattered throughout genomic DNA has been exploited in the identification of polymorphic markers (2,3). Micro-satellite probes have been used to detect polymorphisms in length of restriction fragments (2) and PCR products (3). Variable length "short tandem repeats" (STRs) such as (CA)n are highly polymorphic and serve as informative markers (4,5).
Another recent advance in polymorphic marker analysis is single short primer PCR, or "random amplified polymorphic DNA" (RAPD) marker analysis. Conduct of PCR with genomic DNA using single short (8-10 mer) primers of arbitrary sequence generates a product that can be used in gel electrophoretic fingerprint analysis to generate numerous polymorphic markers (6,7). Although variable number tandem repeat markers, short tandem repeats and RAPD markers have significantly increased the rate of polymorphic marker discovery and the throughput of polymorphic marker analysis, their analysis is limited by the requirement of labor intensive gel electrophoresis, which typically requires several hours of time and accommodates a relatively small number of tests at one time (less than 100).
Microbial identification is another analytical task that benefits from the present invention. Identification of bacterial, viral and mycotic species, strains and subtypes is a key concern in clinical microbiology, for diagnosis of infectious disease, selection of effective pharmaceutical treatment, and epidemiological investigation of the source and spreading of infectious disease. Microbial identification is also a vital capability in the detection and management of biological warfare agents. Microbial identification is also important in, agricultural, industrial and environmental biomonitoring, for example in the detection of pathogens that reduce agricultural productivity as well as microbes that put nutrients into the soil, in the monitoring of industrial bioprocesses, and in the assessment of biodegradation capacity in soil and waste treatment facilities. Microbial identification typically involves time consuming and expensive culturing and biochemical procedures, as well as costly and complex immunological tests. DNA sequencing and PCR analysis can also be performed to achieve accurate microbial identification and typing, but like current DNA typing procedures, these microbial DNA diagnostic tests require gel electrophoretic analysis, which is time consuming and labor intensive and accommodates a relatively low sample throughput. Analysis of microbial populations, important in environmental and industrial settings, is currently a daunting task, typically requiring extensive culturing and a battery of biochemical tests, supplemented by crude classification by visual inspection. Many of the microbial species in environmental samples are not readily culturable, making detection and identification extremely difficult.
Analysis of gene expression is another area that benefits from the present invention. Transcriptional profiling, i.e., analysis of the relative abundance of messenger RNA transcribed from different genes, is critical to the understanding of patterns of gene expression that are associated with all biological processes, including development, differentiation, response to environmental stresses, and other cellular and organismal functions of interest to basic scientists. The ability to analyze patterns of gene expression can lead to discovery of new genes associated with biological processes. A detailed understanding of gene regulation at the transcriptional level is also a premier concern of the pharmaceutical industry, enabling identification of genetic targets for drug development and leading to the understanding of the well known heterogenity in the way different individuals respond to pharmaceutical interventions. Transcriptional profiling is currently conducted by the techniques of "differential display" (Liang, P. and Pardee, A. B. (1992) Science 257:967-971; Liang, et al., (1994) Nucl. Acids Res. 22:5763-5764; Prashar, Y. and Weissman, S. M. (1996) Proc. Nat'l. Acad. Sci., U.S.A. 93:659-663.) and "representational difference analysis" (Hubank, M. and Schatz, D. G. (1994) Nucl. Acids Res. 22:5640-5648; Lisitsyn, N. A. (1995) Trends Genet. 11:303-307), both of which involve PCR, gel electrophoretic analysis of DNA fragments, and a variety of other complex manipulations. A need clearly exists for new technology that enables more robust, rapid and cost effective quantitation of a very large number of gene transcripts.
The prior art is deficient in the lack of effective means for the rapid, simultaneous analysis of a large number of DNA markers, for rapid identification of species, strains, and sub-types and gender, and for rapid transcriptional profiling. The present invention fulfills this longstanding need and desire in the art.